Ana I. Dueñas

Lipopolysaccharide and Sphingosine-1-Phosphate Cooperate To Induce Inflammatory Molecules and Leukocyte Adhesion in Endothelial Cells

Given that TLRs and sphingosine-1-phosphate (S1P) are key players in inflammation, we explored the potential interplay between TLRs and S1P in the adhesion/inflammatory pathways in primary human endothelial cells. As determined by Western blot and flow cytometry, cells treated with LPS (a TLR4 ligand) and S1P showed significantly enhanced expression of adhesion molecules such as ICAM-1 and E-selectin compared with the effect of either ligand alone. Cell-type differences on E-selectin upregulation were observed. In contrast, no cooperation effect on ICAM-1 or E-selectin was observed with a TLR2/TLR1 ligand. Consistent with an increase in adhesion molecule expression, endothelial cell treatment with LPS plus S1P significantly enhanced adhesion of PBMCs under shear stress conditions compared with the effect of either ligand alone and exhibited comparable levels of cell adhesion strength as those after TNF-α treatment. Moreover, LPS and S1P cooperated to increase the expression of proinflammatory molecules such as IL-6, cyclooxygenase-2, and prostacyclin, as determined by ELISA and Western blot. The analysis of signaling pathways revealed the synergistic phosphorylation of ERK upon LPS plus S1P treatment of HUVEC and human aortic endothelial cells and cell-type differences on p38 and NF-κB activation. Moreover, pharmacological and small interfering RNA experiments disclosed the involvement of S1P1/3 and NF-κB in the cooperation effect and that cell origin determines the S1P receptors and signaling routes involved. Sphingosine kinase activity induction upon LPS plus S1P treatment suggests S1P– Sphingosine kinase axis involvement. In summary, LPS and S1P cooperate to increase proinflammatory molecules in endothelial cells and, in turn, to augment leukocyte adhesion, thus exacerbating S1P-mediated proadhesive/proinflammatory properties.

A New Pharmacological Effect of Salicylates: Inhibition of NFAT-Dependent Transcription

The anti-inflammatory effects of salicylates, originally attributed to inhibition of cyclooxygenase activity, are currently known to involve additional mechanisms. In this study we investigated the possible modulation by salicylates of NFAT-mediated transcription in lymphocytic and monocytic cell lines. RNase protection assays showed that 2-acetoxy-4-trifluoromethylbenzoic acid (triflusal) inhibited, in a dose-dependent manner, mRNA expression of several cytokine genes, most of which are NFAT-regulated and cyclosporin A (CsA)-sensitive. In Jurkat cells, the expression of IL-3, GM-CSF, TNF-α, TGF-β1, IL-2, lymphotactin, MIP-1α, and MIP-1β was inhibited to different extents. In THP-1 cells, inhibition of the expression of M-CSF, G-CSF, stem cell factor, IFN-γ, TNF-α, TGF-β1, lymphotoxin-β1, MIP-1α, MIP-1β, and IL-8 was observed. Sodium salicylate and aspirin only showed significant effects at 5 mM. The transcriptional activity of two genes that contain NFAT sites, a GM-CSF full promoter and a T cell-specific enhancer from the IL-3 locus, was also inhibited by salicylates. Transactivation experiments performed with several NFAT-dependent and AP-1-dependent reporter genes showed that triflusal strongly inhibited NFAT-dependent transcription at concentrations as low as 0.25 mM. Sodium salicylate and aspirin were less potent. The triflusal inhibitory effect was reversible and synergized with suboptimal doses of CsA. Experiments to address the mechanism of action of salicylates in the NFAT activation cascade disclosed a mechanism different from that of CsA, because salicylates inhibited DNA-binding and NFAT-mediated transactivation without affecting phosphorylation or subcellular localization of NFAT. In summary, these data describe a new pharmacological effect of salicylates as inhibitors of NFAT-dependent transcription.

Selective attenuation of Toll-like receptor 2 signalling may explain the atheroprotective effect of sphingosine 1-phosphate

AIMS Vascular inflammation is a major atherogenic factor and Toll-like receptor (TLR) 2 ligands, including bacterial and serum lipoproteins, seem to be involved in atherogenesis. On this basis, we analysed the effect of lipoproteins and different lipid components on TLR2-dependent signalling. METHODS AND RESULTS In TLR2-transfected human embryonic kidney 293 cells and human monocytes, oxidized low-density lipoproteins inhibited nuclear factor (NF)-kappaB-driven transcriptional activity and chemokine gene expression in response to TLR2 ligands. Sphingosine 1-phosphate (S1P) and oxidized palmitoyl-arachidonoyl-phosphatidylcholine, but not lipoprotein-carried lysophospholipids, inhibited TLR2 activation. Silencing experiments in TLR2-transfected 293 cells showed that the S1P-mediated attenuation effect is mediated by S1P receptors type 1 and type 2. To address the physiological significance of these findings, additional experiments were performed in human peripheral blood monocytes and monocyte-derived macrophages. In both cell types, S1P selectively attenuated TLR2 signalling, as NF-kappaB and extracellular signal-regulated kinase activation, but not c-Jun amino terminal kinase phosphorylation, were inhibited by physiologically relevant concentrations of S1P. Moreover, the attenuation of TLR2 signalling was partially reverted by pharmacological inhibition of phosphoinositide 3-kinase (PI3K) and Ras pathways. In addition, S1P inhibited the chemokine gene expression elicited by TLR2, but not by TLR4 ligands. CONCLUSION These findings disclose a cross-talk mechanism between lipoprotein components and TLR in which engagement of S1P receptors exert selective attenuation of TLR2-dependent activation via PI3K and Ras signalling. A corollary to these data is that the negative cross-talk of S1P receptors and TLR2 signalling might be involved in the atheroprotective effects of S1P.

Viral and bacterial patterns induce TLR-mediated sustained inflammation and calcification in aortic valve interstitial cells.

BACKGROUND Aortic stenosis shares some ethiopathological features with atherosclerosis and increasing evidence links Toll-like receptors (TLRs) to atherogenesis. METHODS TLR-mediated inflammation and osteogenesis were investigated in human interstitial cells isolated from stenotic and non-stenotic aortic valves. TLR expression and signalling were evaluated by quantitative RT-PCR, flow cytometry, Western blot analysis, ELISA, and cytokine arrays. Osteogenesis was evaluated by measuring alkaline phosphatase activity. RESULTS Interstitial cells from control valves express most TLRs, being TLR4 the most abundant, whereas cells from stenotic valves express higher TLR4 and TLR2 and lower TLR5 and TLR9 transcript levels. When pro-inflammatory pathways were analyzed, we observed that TLR4, TLR2 and TLR3 ligands induced an early activation of NF-κB and p38 MAPK activation in cells from control and stenotic valves. Strikingly, when TLRs sensing viral patterns were studied, a sustained TLR3-mediated activation of NF-κB, a κB-independent induction of catalytically active cyclooxigenase (COX)-2 and ICAM-1 expression, and induction of expression of several chemokines were observed. TLR4, but not TLR2, engagement produced a similar but NF-κB-dependent effect. Moreover, TLR3 and TLR4 agonists induced alkaline phosphatase expression and activity. CONCLUSIONS Exposure of aortic valve interstitial cells to viral and Gram-negative bacteria molecular patterns induces distinct and long-term TLR-mediated pro-inflammatory and pro-osteogenic responses that might be relevant to the pathogenesis of degenerative aortic stenosis.

Varicose Veins Show Enhanced Chemokine Expression

OBJECTIVES Leucocyte infiltration in the wall of varicose veins has been reported previously. This study was designed to investigate the expression of pro-inflammatory cytokines and chemokines in control and in patients with varicose veins and to test the effect of treating varicose vein patients with acetylsalicylic acid (ASA) on cytokine expression prior to removal of varices. MATERIAL AND METHODS Sections of vein were removed during operation from both patient groups, and ribonuclease protection assays (RPAs) were performed to assess the expression of chemokines. Group I included non-varicose saphenous veins from healthy patients undergoing amputation for trauma. Varicose veins were obtained from patients with primary varicose undergoing surgical treatment who received no drug (group II) or treatment with 300 mg day(-1) of ASA for 15 days before surgery (group III). RESULTS Non-varicose veins constitutively expressed low levels of monocyte-chemoattractant protein (MCP-1) and interleukin (IL)-8 mRNA. Varicose veins had a distinct chemokine expression pattern, since significant up-regulation of MCP-1 and IL-8 and a marked expression of IP-10, RANTES, MIP-1alpha and MIP-1beta mRNA were detected. Removal of the endothelium did not alter this pattern. Varicose veins obtained from patients treated with ASA showed a consistent decrease in chemokine expression, although it did not reach statistical significance. CONCLUSIONS Varicose veins showed increased expression of several chemokines compared to control veins. A non-significant reduction of activation was observed following treatment with ASA for 15 days.

Serologic evidence of human infection by Francisella tularensis in the population of Castilla y León (Spain) prior to 1997

Prior to an outbreak in Castilla y León in December 1997, tularaemia was practically non-existent in Spain. In this paper we studied the prevalence of antibodies against Francisella tularensis in a representative sample of the population (4825 people) from Castilla y León (Spain) in samples collected before this outbreak. Antibodies against F. tularensis were detected in nine (0.19%) of the 4825 sera, with antibody titres ranging from 1/20 to 1/160. Of these nine sera, one was positive in seroagglutination against Brucella. Seroagglutination against other bacteria (Yersinia enterocolitica O:9 and O:3 and Proteus OX19) was negative in all sera. Seroprevalence of antibodies in females was 0.20% and 0.17% in males; no statistically significant differences were found in prevalence in terms of sex, age or province.

Seroprevalencia de infección por Echinococcus granulosus en la población de Castilla y León

Introduccion La hidatidosis es una de las zoonosis mas importantes en la Comunidad Autonoma de Castilla y Leon. Este estudio pretende conocer la seroprevalencia de infeccion por Echinococcus granulosus en dicha comunidad autonoma. Metodos Se han estudiado 4.824 muestras de suero pertenecientes a 4.824 personas seleccionadas de forma aleatoria y que constituian una muestra representativa de la poblacion de las provincias de Castilla y Leon. En cada suero se estudio la presencia de anticuerpos de clase IgG frente a Echinococcus granulosus mediante una prueba de enzimoinmunoanalisis indirecto de fabricacion propia. Resultados Se detectaron anticuerpos de clase IgG frente a Echinococcus granulosus en el 3,40% de los sueros estudiados (164 positivos de 4.824), oscilando entre el 1,26 y el 7,10% segun la provincia de origen. La seroprevalencia de anticuerpos aumentaba significativamente con la edad. No se observaron diferencias estadisticamente significativas entre las seroprevalencias halladas en mujeres y en varones (3,14% frente a 3,66%). Conclusion La seroprevalencia de infeccion por E. granulosus en la Comunidad Autonoma de Castilla y Leon es todavia alta. Estos datos de seroprevalencia contribuyen a la vigilancia de la hidatidosis dentro de un programa control de esta enfermedad.

Diagnóstico de laboratorio y evolución serológica de pacientes con tularemia

Fundamento La tularemia era una enfermedad practicamente inexistente en Espana hasta finalesde 1997, cuando se declaro un brote epidemico en nuestra comunidad. El objetivo denuestro trabajo ha sido estudiar los datos existentes sobre el diagnostico microbiologico de 55pacientes que sufrieron tularemia. Pacientes Y Metodos Se obtuvieron 32 muestras para cultivo pertenecientes a 19 pacientes y151 sueros correspondientes a 55 pacientes. El diagnostico serologico se realizo mediante seroaglutinacionen tubo y microaglutinacion. En todos los sueros se realizo una seroaglutinacionde Wright (SAW) y un test de Coombs frente a Brucella y seroaglutinaciones frente a Yersiniaenterocolitica O:9, Yersinia enterocolitica O:3 y Proteus OX 19. Resultados Se aislo F. tularensis en dos muestras (6,25%) de las 32 estudiadas. Se obtuvierontitulos mayores o iguales a 1/160 en el 78,2% y en el 74,5% de los sueros iniciales por seroaglutinacionen tubo y microaglutinacion, respectivamente. La correlacion entre las dos pruebasfue de 0,80 (p Conclusiones El cultivo de F. tularensis es poco sensible. La correlacion obtenida entre la seroaglutinacionen tubo y microaglutinacion es buena. Ambas tecnicas son utiles en el diagnosticode la tularemia, con algunas ventajas de la microaglutinacion sobre la aglutinacion. Background Tularemia was practically unknown in Spain until the end of 1997, when an epidemicoutbreak was declared. This paper presents the data on microbiological diagnosis of 55patients who suffered from tularemia. Patients and Methods Thirty-two samples from 19 patients and 151 serum samples from 55 patientswere obtained for culture. Serologic diagnosis was performed by tube seroagglutinationand microagglutination. Three types of tests were performed on all sera: Wright seroagglutination(WSA), Coombs test against Brucella spp. and seroagglutination against Yersinia enterocoliticaO:3, Yersinia enterocolitica O:3, and Proteus OX 19. Results F. tularensis was found in two samples (6.25%) of the 32 received. Titers. 1/160were obtained in 78.2% and 74.5% of the initial sera by tube seroagglutination and microagglutination,respectively. Correlation between the two tests was 0.80 (p Conclusions Culture of F. tularensis has low sensitivity. The correlation obtained between tubeseroagglutination and microagglutination is good. Both techniques are useful in routine diagnosisof tularemia, although microagglutination has some advantages over tube agglutination.

Synergy between sphingosine 1-phosphate and lipopolysaccharide signaling promotes an inflammatory, angiogenic and osteogenic response in human aortic valve interstitial cells.

Given that the bioactive lipid sphingosine 1-phosphate is involved in cardiovascular pathophysiology, and since lipid accumulation and inflammation are hallmarks of calcific aortic stenosis, the role of sphingosine 1-phosphate on the pro-inflammatory/pro-osteogenic pathways in human interstitial cells from aortic and pulmonary valves was investigated. Real-time PCR showed sphingosine 1-phosphate receptor expression in aortic valve interstitial cells. Exposure of cells to sphingosine 1-phosphate induced pro-inflammatory responses characterized by interleukin-6, interleukin-8, and cyclooxygenase-2 up-regulations, as observed by ELISA and Western blot. Strikingly, cell treatment with sphingosine 1-phosphate plus lipopolysaccharide resulted in the synergistic induction of cyclooxygenase-2, and intercellular adhesion molecule 1, as well as the secretion of prostaglandin E2, the soluble form of the intercellular adhesion molecule 1, and the pro-angiogenic factor vascular endothelial growth factor-A. Remarkably, the synergistic effect was significantly higher in aortic valve interstitial cells from stenotic than control valves, and was drastically lower in cells from pulmonary valves, which rarely undergo stenosis. siRNA and pharmacological analysis revealed the involvement of sphingosine 1-phosphate receptors 1/3 and Toll-like receptor-4, and downstream signaling through p38/MAPK, protein kinase C, and NF-κB. As regards pro-osteogenic pathways, sphingosine 1-phosphate induced calcium deposition and the expression of the calcification markers bone morphogenetic protein-2 and alkaline phosphatase, and enhanced the effect of lipopolysaccharide, an effect that was partially blocked by inhibition of sphingosine 1-phosphate receptors 3/2 signaling. In conclusion, the interplay between sphingosine 1-phosphate receptors and Toll-like receptor 4 signaling leads to a cooperative up-regulation of inflammatory, angiogenic, and osteogenic pathways in aortic valve interstitial cells that seems relevant to the pathogenesis of aortic stenosis and may allow the inception of new therapeutic approaches.

Prevalencia de anticuerpos frente a Francisella tularensis en la población de Castilla y León con anterioridad a 1997

Fundamento y objetivos: Este trabajo pretende conocer la prevalencia de infeccion por Francisella tularensis en la poblacion de Castilla y Leon previa al brote de tularemia humana de finales de 1997. Sujetos y metodo: Se obtuvieron 4.825 sueros (entre abril de 1996 y abril de 1997) de residentes en Castilla y Leon. Se realizo una prueba de microaglutinacion en placa para detectar anticuerpos anti-F. tularensis. En los sueros positivos se llevaron a cabo seroaglutinaciones en tubo frente a Brucella, Yersinia enterocolitica y Proteus. Resultados: Se detectaron anticuerpos anti-F. tularensis en 9 (0,19%) de los 4.825 sueros. De esos 9 sueros, uno fue positivo en la seroaglutinacion frente a Brucella, siendo todos negativos frente a las otras bacterias. Conclusiones: Antes de 1997 la seroprevalencia de anticuerpos anti-F. tularensis en la poblacion de Castilla y Leon era baja (0,19%).